pGLO Bacterial Transformation

1. DNA RNA Protein Trait pGLO Bacterial Transformation

2. SAFETY FIRST! • WEAR GLOVES • DO NOT OPEN PLATES • CLOROX LAB STATION WHEN DONE • BRING PLATES TO BISHOP TO BE DESTROYED. • WASH HANDS WITH SOAP – HAPPY BIRTHDAY JUMP TO SLIDE 29

3. What was the point of this lab?

4. What was alive? Escherichia coli, a bacterium Glass E. coli by Luke Jerram

5. Escherichiacoli (E.coli) • Found in human large intestines • Model organism for molecular biology • model organism – species that has been extensively studied to better understand biological phenomena and give insight to workings of other organisms • “White rat” of molecular biology light microscope scanning electron microscope

6. What was the source of “new genes”? A plasmid, genetically engineered to carry specific genes

7. Plasmid • commonly found as small circular, double-stranded DNA molecules in bacteria; a NORMAL component of prokaryotes (and a few eukaryotes!) • physically separate from, and replicate independently of, chromosomal DNA within a cell • carry genes that may benefit survival of the organism (e.g. antibiotic resistance) • may be modified to express proteins of interest; these “man-made” plasmids are widely used as vectors in molecular cloning

8. Bacterial DNA DNA in nucleoid region – NO nucleus Bacteria have a single circular chromosome Bacteria have accessory DNA, small circular pieces called plasmids Bacterial cell Plasmid DNA Genomic DNA

9. pGLO, a recombinant plasmid & vector • Recombinant plasmid– plasmid into which DNA fragments or genes have been inserted • Vector – plasmid used experimentally as tool to clone, transfer, and manipulate genes • Because bacteria divide rapidly, they can be used as “factories” to copy DNA fragments in large quantities

10. How did you get the plasmid intothe E. coli? TRANSFORMATION - Process by which bacteria take up plasmids from the environment and express the plasmid genes EXPRESS – make proteins TRANSFORMATION IS A NATURAL PROCESS BUT SOME BACTERIA ARE “BETTER” AT IT THAN OTHERS. Our procedure was developed to improve the likelihood E. coli will transform.

11. Bacterial TransformationProcess by which bacteria take up plasmids from the environment and express the plasmid genes Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

12. Transformation Procedure • Suspend bacterial colonies in Transformation solution • Add pGLO plasmid DNA • Place tubes on ice • Heat-shock at 42°C and place on ice • Incubate with nutrient broth • Streak plates: LB LB/amp/+ LB/amp/- LB/amp/ara/+

13. Reasons for Performing Each Transformation Step? Ca++ O Ca++ O P O Base • Transformation solution = CaCI2 Positive charge of Ca++ ions shields negative charge of DNA phosphates O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH

14. Why Perform Each Transformation Step? Cell wall GFP 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance)

15. What is Nutrient Broth? • Luria-Bertani (LB) broth • Medium that contains nutrients for bacterial growth and gene expression • Carbohydrates • Amino acids • Nucleotides • Salts • Vitamins

16. How do you know which cells transformed (took in plasmid)? pGLO has reporter genes • bla (ampr) • GFP • araC

17. 3 Genes of interest in pGLO plasmid This is DNA! • Gene for Beta Lactamase (bla) • Protein that inactivates the antibiotic ampicillin • Gene for Green Fluorescent Protein (GFP) • Aequoreavictoria jellyfish gene • Proteinfluoresces green after absorbing UV or blue light • Gene for araCregulator protein • Protein that regulates GFP transcription – “turns GFP gene on”

18. ANY QUESTIONS?

19. STUDENT MANUAL • Lesson 1 page 33 • 1, 2, 4 – you should be able to answer • 3 safety ideas? • Does not produce toxic compounds which make people sick • Grows well in lab but can’t survive outside lab • Not able to infect animals and plants

20. STUDENT MANUAL • Lesson 1 page 34 • Phenotype – physical appearance • Starter Plate: Let’s answer the questions together.

21. STUDENT MANUAL Number of colonies? What is a colony? Descendants of a single cell!

22. STUDENT MANUAL Size of of colonies?

23. STUDENT MANUAL Color of colonies?

24. STUDENT MANUAL Distribution of colonies?

25. STUDENT MANUAL Appearance in UV light?

26. STUDENT MANUAL Ability to reproduce in presence of antibiotic?

27. STUDENT MANUAL You should be able to answer questions 1 and 2

28. STUDENT MANUAL • Lesson 2 page 42 • 1? You Explain • 2? You Explain • 3? You Explain • 4? You have 2 Control plates – You Explain

29. STUDENT MANUAL • Lesson 3 page 43 • 1, 2, 3, 4 You do. • But first let’s predict!

30. PREDICTION RESULTS Did bacteria get plasmid? Is bacteria ampicillin resistant? Will bacteria glow in the dark?

31. PREDICTION RESULTS Did bacteria get plasmid? Is bacteria ampicillin resistant? Will bacteria glow in the dark?

33. STUDENT MANUAL • Lesson 3 page 44 • 1, 2 You do • 3 Badly worded. Infer cells are expressing the bla gene – making a protein that inactivates ampicillin • 4 Hint what plates should you compare and why?

34. STUDENT MANUAL • Lesson 3 page 45 • 1 The sample did not flouresce • 2, 3 You do • 4 You must answer based on you results If successful, state evidence from plates If unsuccessful, state evidence from plates

35. STUDENT MANUAL • Lesson 3 page 46 • Yes, E. coli grow on the LB plate • 1, 2 you answer • 3 a. What are the 2 factors? • 3 b. What does arabinose do? What does UV light do • 3 c. You answer

36. STUDENT MANUAL • Lesson 4 follow the instructions and do the math. • Page 48 #1. Some of you have no colonies on the LB/amp/ara/+pGLO plate USE THIS AS NUMBER OF COLONIES: 50 PAGE 51: REPORT TO BISHOP AS SOON AS YOU HAVE NUMBERS. WE WILL SHARE NUMBERS LATER.

38. LB/Amp LB/Amp/Ara LB Grow?Glow? • Follow protocol • On which plates will colonies grow? • Which colonies will glow?

39. From Wikipedia, the free encyclopedia Jump to: navigation, search Left: pGLO under ambient light Right: pGLO visualized under ultraviolet light The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequoreavictoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates. pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows: -Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family) -araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose) -GFP, the green fluorescent protein Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The pGLO plasmid was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba. Other features on pGLO, like most other plasmids, include: a selectable marker, Ori (origin of replication), and an MCS (multiple cloning site) located at the end of the GFP gene. The plasmid is 5371 base pairs long. In supercoiled form, it runs on an agarose gel in the 4200-4500 range.[1][2][3]

40. The green fluorescent protein GFP consists of 238 amino acids, linked together in a long chain. This chain folds up into the shape of a beer can. Inside the beer can structure the amino acids 65, 66 and 67 form the chemical group that absorbs UV and blue light, and fluoresces green. (Credit: Image courtesy of Nobel Foundation)

42. Links to Real-world • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms

44. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

45. Transformation Procedure Overview Day 2 Day 1

46. What is a plasmid? • A circular piece of autonomously replicating DNA • Originally evolved by bacteria • May express antibiotic resistance gene or be modified to express proteins of interest

47. What is Transformation? • Uptake of foreign DNA, often a circular plasmid GFP Beta-lactamase Ampicillin Resistance

48. Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid

49. ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D LacI Z Y A RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation

50. ara GFP Operon ara Operon araC GFP Gene araC B A D Effector (Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation